Polycyclic aromatic hydrocarbons, such as benzo[a]pyrene (BaP) and benzo[c]phenanthrene (BcPh), are widespread environmental pollutants, many of which are potent carcinogens in mammals. These hydrocarbons are metabolically activated through the action of cytochrome P450 and epoxide hydrolase to give highly reactive diol epoxides (DEs). A plausible mechanism for the induction of cell damage by DEs involves opening of the epoxide ring by the exocyclic amino groups of the purines in DNA, followed by erroneous replication of the modified residues, leading to mutations. Sixteen DE-purine adducts are possible for a given parent hydrocarbon. Duplex oligonucleotides containing these adducts exhibit distinctly different structural motifs which depend on the hydrocarbon, the target purine nucleoside (dA or dG) and the stereochemistry of the adduct, and thus provide unique tools to probe the catalytic and recognition sites of DNA-processing enzymes. We have developed new synthetic methods which make possible the relatively facile synthesis of adducted nucleosides and site-specifically adducted oligonucleotides on a scale suitable for NMR and X-ray crystallographic studies. As a result we now report the first crystal structures of a DE-modified nucleoside derivative and of a DE adduct in an oligonucleotide bound to a DNA polymerase. Crystal Structures. Although nucleoside adducts of DEs had previously been recalcitrant to crystallization, we recently succeeded in crystallizing an O6-allyl derivative of a BaP DE-dG adduct for X-ray structural determination (4). This adduct derives from trans opening at C10 of the (-)-(7S,8R)-diol (9R,10S)-epoxide enantiomer by the exocyclic N2-amino group of dG. The crystal structure provides independent and unambiguous proof of the absolute configuration of the adduct based on the spatial relationship between the chiral carbon atoms of the sugar and the four asymmetric centers of the hydrocarbon moiety. In the crystal, the spatial orientations of the purine and pyrene ring systems as well as the conformation of the partially saturated ring are in close agreement with deductions based on NMR and circular dichroism measurements in solution. The modified dG in the crystal exhibits a syn glycosidic torsion angle, a conformation that is unusual in B-DNA but which may be involved in error-prone nucleotide incorporation opposite DE-adducted purines by DNA polymerases. This year we have also determined the first crystal structure of a DE-modified oligonucleotide bound at the active site of a DNA polymerase, Dpo4 (5). Dpo4 is an archaeal enzyme belonging to the Y-family of relatively error-prone polymerases which are able to bypass bulky adducts that normally block replicative DNA polymerases. In the crystal structure, Dpo4 was bound to an oligonucleotide template containing a dA adduct derived from cis opening of the carcinogenic (+)-BaP (7R,8S)-diol (9S,10R)-epoxide with a dT positioned in the primer opposite the adduct and with an incoming dATP corresponding to translesion synthesis beyond the modified base pair. Two conformations of the adduct in the template-primer were observed in each unit cell: one in which the aromatic portion of the hydrocarbon is stacked with the modified adenine-thymine base pair, and one in which the hydrocarbon occupies an extrahelical position in the nascent major groove. The latter conformation exhibits the more favorable geometry for primer extension by the incoming dATP, although NMR structures indicate that analogous BaP DE-dA adducts in fully-duplex DNA prefer an intercalated conformation. Biochemical Studies. Enzymes investigated this year have included vaccinia virus topoisomerase I, HIV-1 integrase and human mitochondrial DNA polymerase gamma. i) The first step in the mechanism whereby vaccinia topoisomerase I relaxes supercoiled DNA involves making a transient single-strand break in duplex DNA to give a phosphotyrosyl bond between the enzyme and a DNA 3'-phosphate group, with expulsion of a DNA fragment containing a free 5'-hydroxyl group. As described in last year?s Report, topoisomerase-substrate contacts in the enzyme?s 5'-C+5-C+4-C+3-T+2-T+1-3' recognition sequence as well as at the adjacent cleavage site (T+1/A-1) were mapped by use of minor-groove-bound and intercalated adducts derived from BaP DEs, which were incorporated into the complementary, nonscissile strand. These investigations have now been extended (7) to BcPh DE adducts at dA and dG residues, which intercalate from the major and minor grooves, respectively. A BcPh DE adduct intercalated at the T+1/A-1 cleavage site decreased the rate of topoisomerase cleavage by a factor of 450, whereas adducts 3' to the cleavage site had smaller effects. BcPh DE dA or dG adducts intercalated between base pairs at positions +4 to +1 of the recognition sequence dramatically reduced the rate (>2000-fold) or abolished all cleavage, whereas dG adducts intercalated between the +4 and +5 positions of the recognition sequence had little effect. These results are consistent with our previous observations concerning the importance of enzyme-substrate contacts at these positions in the recognition sequence, and suggest that the mode of intercalation (from the major or the minor groove) does not critically affect the inhibitory action of these hydrocarbon adducts. ii) We have also explored the effects of BaP DE-dG adducts on DNA cleavage and strand transfer reactions catalyzed by HIV-1 integrase (3). These adducts inhibited cleavage when located in the minor groove near the enzyme?s specific cleavage site (5'-A/GT-3') at the 3'-end of a synthetic oligonucleotide substrate. Adducts at integration sites inhibited strand transfer and resulted in integration at positions distal to the ?normal? integration sites. iii) Replication of oligonucleotides containing site-specific BaP and BcPh DE adducts by an exonuclease-deficient form of human mitochondrial DNA polymerase gamma has been examined (1). These DE adducts resulted largely in purine misincorporation opposite the adducts as well as extensive blockage of translesion synthesis beyond the adduct site.